L9. Proteins. Dialysis.

Electrophoresis

Properties of proteins
1. Molecular Weight - the proteins generally have large molecular weights ranging
between 5 × 103 and 1 × 106 Da. It might be noted that the values of molecular
weights of many proteins lie close to or multiples of 35,000 and 70,000 Da.
2. Colloidal Nature - because of their giant size, the proteins exhibit many colloidal
properties. Their diffusion rates are extremely slow and they may produce
considerable light-scattering in solution, thus resulting in visible turbidity (Tyndall
effect).
3. Denaturation - denaturation refers to the changes in the properties of a protein. In
other words, it is the loss of biologic activity. In many instances the process of
denaturation is followed by coagulation - a process where denatured protein
molecules tend to form large aggregates and to precipitate from solution.
4. Amphoteric Nature - like amino acids, the proteins are amphoteric, i.e., they act as
acids and alkalies both. These migrate in an electric field and the direction of
migration depends upon the net charge possessed by the molecule. The net charge is
influenced by the pH value. Each protein has a fixed value of isoelectric point (pl) at
which it will move in an electric field.The pH at which a protein has equal number of
positive and negative charges is known as isoelectric pH. When subjected to an
electric field the proteins do not move either towards anode or cathode, hence this
property is used to isolate proteins. The proteins become least soluble at pI and get
precipitated.
5. Ion Binding Capacity - the proteins can form salts with both cations and anions
based on their net charge.
6. Solubility - the solubility of proteins is influenced by pH. Solubility is lowest at
isoelectric point and increases with increasing acidity or alkalinity. This is because
when the protein molecules exist as either cations or anions, repulsive forces between
ions are high, since all the molecules possess excess charges of the same sign. Thus,
they will be more soluble than in the isoelectric state.
7. Optical Activity - all protein solutions rotate the plane of polarized light to the left,
i.e., these are levoratotory.

Dialysis is a separation method of a mixture of substances, based on different

diffusion capacity of the mixture components, through a semipermeable membrane.
The membranes represent structures that delimitate two compartments (or many). In a
simple way, the membrane can be imagined as a surface -barrier at the interface of two
liquids. But membrane is also a connection bridge between the compartments of the phases it
separates (due to its permeability) and meantime is the place of complex activities related to
the transport phenomena.
Dialysis is used to remove lower-molecularcomponents from protein solutions, or to
exchangethe medium. Dialysis is based on thefact that due to their size, protein moleculesare
unable to pass through the pores of asemipermeable membrane, while lower-
molecularsubstances distribute themselvesevenly between the inner and outer spacesover
time. After repeated exchanging of theexternal solution, the conditions inside thedialysis tube
(salt concentration, pH, etc.)will be the same as in the surrounding solution.
 interior of the dialysis bag exterior of the dialysis bag
Principle of dialysis

Materials used for dialysis membranes must be in a film form, or substances with a
high polymerization grade, which swell in the presence of solvents and are transformed in
molecular sieves, whose pores permit the crossing of substances (including the solvents) with
low molecular weight, hindering the crossing of the components with high molecular weight
(proteins).
The membrane must be chemically inert to avoid the binding of the components to the
membrane. Now, the membranes are made of cellophane (cellulose) obtained with special
procedures.
The dialysis method, applied to volumes of 1-100 milliliters can be realized using the
following procedure: the solution to be dialyzed is closed into a bag, having properties of
semipermeable membrane, and is suspended in a much bigger volume of solvent, in a vessel
maintained at a certain temperature. Dialysis, being a diffusing controlled process, the
passing of some substances from the bag into the solvent will increase their concentration in
the exterior. The mixing is needed to dissipate the diffused substances in the solvent,
reducing their concentration on the external surface of the membrane, and avoiding the
formation of a concentration gradient.
The effect of dialysis has been used to realize an extrabody device, called „artificial
kidney”, used to purify the blood of components with low molecular weight that are normally
excreted in the urine.

Electrophoresis is a motion of dispersed particles relative to a fluid under influence

of electric field that is space uniform.
Electrophoresis occurs because particles dispersed in fluid carry electric surface
charge, practically always. Electric field exerts electrostatic Coulomb force on the particle
through these charges.
The migration of proteins, in an electrical field, is due to their amphoteric nature,
which permits an electric charge dependent on the pH of the solution:
IsoelectricpH
H+ HO-
H 3N +
CH COOH H 3 N+ C H C O O - H 2N CH COO-
R R R
Cationic form Amphionic form Anionic form

The electrophoretic migration speed depends on the following factors:

- the electrical charge of the molecule
- the volume and the form of the molecule
- the applied tension of the electrical field
- the nature of the used solid support
- the work temperature
 The electrophoretic mobility is directly proportional to the charge of the protein that
migrates under the action of the electric field and conversely proportional to the molecule
size and medium viscosity (the buffer used in electrophoresis).
There are two types of electrophoresis, considering the electrophoretic medium
nature:
- Electrophoresis in solution – the particle’s migration takes place in solution.
This system, elaborated by Tiselius, has a low resolution power and a high
consume of materials. It is not used in analytical purposes but only for the
measurement of the electrophoretic mobility.
- Zonal electrophoresis – the water solution, containing the electrical charged
particles, is applied on a solid material or matrix. A net delimitation, as strips, of
the separated particles (i.e. proteins) is obtained.
Zonal electrophoresis is the most spread electrophoretic technique used in
biochemistry, specially for the analysis of proteins in serum, urine, cerebrospinal fluid, as
proteins from erythrocytes, tissues, etc.
As s function of the solid material used in the process, there are different variants:

a.Filter paper electrophoresis. Is a less used technique due to a low resolution and
the long time needed for separation. Also, the electrophoretic separation is associated to a
chromatographic effect due to the absorption of the molecules by the hydroxyl groups of
cellulose, effect that decreases the migration speed and affects the particle separation.

b. Electrophoresis on cellulose acetate. The method is improved compared to the

previous one because the majority of the hydroxyl groups in cellulose has been acetylated;
the molecule’s absorption on the electrophoretic support is reduced, that permits a superior
resolution and speed of migration. Also, the cellulose acetate presents other advantages as:
- It needs smaller quantities of the analyzed material.
- It is transparent, so it permits the direct quantification by spectrophotometry of the
separated strips.
- It can be dissolved in different solvents, that permits a easier and rapid isolation of
the components.

c. Gel electrophoresis
This type of electrophoresis is a technique used for the separation of DNA, RNA, or
protein molecules. It is usually performed for analytical purposes, but may be used as a
preparative technique to partially purify molecules prior to use of other methods such as mass
spectrometry, PCR, cloning, DNA sequencing, or Southern blotting for further
characterization.
The term "gel" in this instance refers to the matrix used to contain, then separate the
target molecules. In most cases the gel is a crosslinked polymer whose composition and
porosity is chosen based on the specific weight and composition of the target to be analyzed.
When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is
usually composed of different concentrations of acrylamide and a cross-linker, producing
different sized mesh networks of polyacrylamide. When separating larger nucleic acids
(greater than a few hundred bases), the preferred matrix is purified agarose. In both cases, the
gel forms a solid, yet porous matrix. Acrylamide, in contrast to polyacrylamide, is a
neurotoxin and must be handled using Good Laboratory Practices to avoid poisoning.
By placing the molecules in wells in the gel and applying an electric current, the
molecules will move through the matrix at different rates, towards the anode if negatively
charged or towards the cathode if positively charged.
 The most commonly used procedure forchecking the purity of proteins is sodium
dodecylsulphate polyacrylamide gel electrophoresis(SDS-PAGE). In electrophoresis,
moleculesmove in an electrical field.Normally, the speed of their movement dependson three
factors—their size, their shape,and their electrical charge.In SDS-PAGE, the proteinmixture
is treatedin such a way that only the molecules’ massaffects their movement. This is achieved
byadding sodium dodecyl sulphate(C12H25-OSO3Na), the sulfuric acid ester of lauryl alcohol
(dodecyl alcohol). SDS is a detergent withstrongly amphipathic properties. Itseparates
oligomeric proteins into their subunitsand denatures them. SDS moleculesbind to the
unfolded peptide chains (ca.0.4 g SDS / g protein) and give thema stronglynegative charge.
To achieve complete denaturation,thiols are also added in order to cleavethe disulphide
bonds.Following electrophoresis, which is carriedout in a vertically arranged gel of
polymericacrylamide, the separated proteins aremade visible by staining.
In medicine, protein electrophoresis is a method of analyzing a mixture of proteins
by means of agarose or cellulose acetate electrophoresis, mainly in blood serum (plasma is
not suitable).

Schematic representation of a protein electrophoresis gel

Absolute and relative values of serum proteins

Normal values Protein concentration (g%)
Protein fraction
% 7-9 g %
Serum albumin 50 - 60 4.5 – 5.5
Serum globulin 1 3.4 – 5.5 0.3 – 0.8
Serum globulin 2 6.0 – 12.0 0.48
Serum globulin  9.0 – 15.0 0.55 – 1.11
Serum globulin  15.0 - 21 0.64 – 1.28

Experimental part
1. Pipet 2 ml of 1% albumin solution in a dialysis bag, seal it and weigh it using an
electronic balance.
2 dialysis bags are needed!
2. In a beaker, measure 990 ml of distilled water and 10 ml of ammonium sulphate
solution 10%.
3. Put the dialysis bags into the beaker, and then place the beaker on a magnetic
stirrer.
 Identification reactions:
1. Into 3 test tubes, pipet as following:
Reagent (µL) E1 E2 E3
(albumin) (ammonim ions) (sulphate ions)
Biuret reagent 500 µL - -
Nessler reagent - 500 µL -
BaCl2 solution 500 µL
Albumin solution 1% 100 µL - -
Ammonium sulphate solution 10% - 100 µL 100 µL
Identification reactions PURPLE color YELLOW- WHITE
ORANGE precipitate precipitate
2. The first part of the experiment takes 30 minutes.
3. In the meantime, pipet into 6 test tubes, as following: (E4-E5-E6 –to analyze the
solution from the dyalisis bag) and (E7-E8-E9 to analyze the solution from the
beaker):
Reagent (µL) E4 E5 E6 E7 E8 E9
Biuret reagent 500 - - 500
Nessler reagent - 500 - 500
BaCl2 solution 500 500
4. After 30 minutes, take out one dialysis bag and weigh it.
5. Open the dialysis bag and pipet as following:
Reagent (µL) E4 E5 E6 E7 E8 E9
Biuret reagent 500 - - 500
Nessler reagent - 500 - 500
BaCl2 solution 500 500
Solution from the 100 100 100 - - -
dialysis bag
Solution from the - - - 100 100 100
beaker
Identification PURPLE YELLOW- WHITE BLUE YELLOW- WHITE
reactions color ORANGE precipitate color ORANGE precipitate
precipitate precipitate
6. Start again the dialysis process for bag no 2. After 30 minutes, pipet as following:
Reagent (µL) E4 E5 E6 E7 E8 E9
Biuret reagent 500 - - 500
Nessler reagent - 500 - 500
BaCl2 solution 500 500
Solution from the 100 100 100 - - -
dialysis bag
Solution from the - - - 100 100 100
beaker
Identification PURPLE YELLOW- WHITE BLUE YELLOW- WHITE
reactions color ORANGE precipitate color ORANGE precipitate
precipitate precipitate

Conclusions:
1. Albumin (proteins) from the intracellular medium have a high molecular weight
(MW≈ 47000 Da) and can not pass through the semipermeable membrane.
2. Through the semipermeable membrane, the ammonium and sulphate ions will
pass into the extracellular medium.